Food Biochemistry and Food Processing

244 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

use of only calf rennet. A method has been pub-
lished by the International Dairy Federation to de-
tect fermentation-produced chymosin (Collin et al.
2003). This method uses immunochemical tech-
niques (ELISA) to detect such chymosin in rennet
solutions. It cannot be used for cheese.
Thus, traditional calf rennet and rennet substitutes
derived from Mucor Miehei, Cryphonectria, Para-
sitica,and others are still used, especially for high
cooking temperature cheeses such as Swiss and
Mozzarella. Since the introduction of fermentation
chymosin, the cost of traditional calf rennet has
dropped considerably, and they both now cost ap-
proximately the same. Fungal rennets are still avail-
able at approximately 65% of the cost of the recom-
binant chymosin.
A coagulating enzyme ofMucor mieheicloned
intoAspergillus oryzaewas developed by Novo Nor-
disk of Denmark. This product hydrolyzes kappa-
casein only at the 105-106 bond, and reduces protein
losses to whey.

RENNETPRODUCTION BYSEPARATION OF
BOVINEPEPSIN

Traditional calf rennet contains about 5% pepsin and
almost 95% chymosin. Industrial purification of stan-
dard rennet to 100% chymosin rennet by the removal
of pepsin was developed by Sanofi-Bioingredients
Co. The process involves separation of the pepsin by
ion exchange. Chymosin has no charge at pH 4.5, but
bovine pepsin is negatively charged. Traditional com-
mercial calf rennet is passed through an ion-exchange
column containing positively charged ions. Bovine
pepsin, due to its negative charge, is retained, and
chymosin passes through and is collected, resulting
in a 100% chymosin product (Pszczola 1989).

RENNET SUBSTITUTES

Improved farming practices, including improved
genetics, have led over the years to significant
increases in the milk production capacity of dairy
cattle. As a result, while total milk production in the
world has increased, the total cow population has
declined. Hence, there has been a reduction in calf
populations and the availability of traditional calf
rennet. Furthermore, the increased practice of rais-
ing calves to an older age for meat production fur-
ther reduced the numbers available for rennet pro-
duction. It is for these reasons that a shortage of calf

rennet occurred and substitutes were sought. Various

proteolytic enzymes from plant, microbial, and ani-

mal sources were identified and developed for com-

mercial applications. These enzymes should possess

certain key characteristics to be successful rennet sub-

stitutes: (1) the clotting-to-proteolytic ratio should

be similar to that of chymosin (i.e., the enzyme

should have the capacity to clot milk without being

excessively proteolytic); (2) the proteolytic speci-

ficity for beta-casein should be low because other-

wise bitterness will occur in cheese; (3) the substitute

product should be free of contaminating enzymes

such as lipases; and (4) cost should be comparable to

or lower than that of traditional rennet.

ANIMAL

Pepsin derived from swine shows proteolytic activity

between pH 2 and 6.5, but by itself it has difficulty in

satisfactorily coagulating milk at pH 6.6. For this

reason, it is used in cheese making as a 1:1 blend

with rennet. Pepsin, used alone as a milk coagulator,

shows high sensitivity to heat and is inclined to cre-

ate bitter cheese if the concentrations added are not

calculated and measured exactly (Kosikowski and

Mistry 1997).

PLANT

Rennet substitutes from plant sources are the least

widely used because of their tendency to be exces-

sively proteolytic and to cause formation of bitter fla-

vors. Most such enzymes are also heat stable and

require higher setting temperatures in milk. Plant

sources include ficin from the fig tree, papain from

the papaya tree, and bromelin from pineapple. A not-

able exception to these problems is the proteolytic en-

zymes from the flower of thistle(Cynara cardunacu-

lus),as reported by Vieira de Sa and Barbosa (1970)

andCynara humilis(Esteves and Lucey 2002). These

enzymes have been used successfully for many years

in Portugal to make native ripened Serra cheese with

excellent flavor and without bitterness.

In India, enzymes derived fromWithania coagulans

have been used successfully for cheese making, but

commercialization has been minimal, especially with

the development of genetically derived chymosin.

MICROBIAL

Substitutes from microbial sources have been very

successful and continue to be used. Many act like